There is a trend toward widespread testing of blood and other body-fluid analytes which are predictive of health conditions, such as risk of coronary disease. In some assays, such as for determination of serum lipoproteins which are predictive of risk of coronary disease, the analyte of interest, either high-density lipoproteins (HDL) or low-density lipoproteins (LDL), must be separated from other lipoproteins present in the sample fluid.
Considering particularly the case of lipoprotein assays, there are four major classes of lipoproteins found in plasma: chylomicrons, very-low density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Chylomicrons are large triglyceride-rich particles synthesized in the intestines and serve to transport dietary fats into the circulation. The process of synthesis, secretion, and clearance of chylomicrons from the blood is usually complete within 5-6 hours after a meal, and these particles are usually not detected in serum after a longer period of fasting.
VLDL are triglyceride-rich lipoproteins which are synthesized in the liver and ultimately converted to LDL, which transports most of the plasma cholesterol in humans. HDL is involved in the catabolism of triglyceride-rich lipoproteins, and in the removal of cholesterol from peripheral tissues, and transport to the liver.
Numerous investigations indicate that LDL is an important causative agent of coronary heart disease and other atherogenic conditions, and that high levels of serum cholesterol associated with LDL are indicative of increased risk of atherogenic disease. An inverse relationship between serum HDL levels and risk of coronary disease has also been established. In particular, if the proportion of serum cholesterol associated with HDL is low, the risk of coronary disease is increased.
In view of the importance of HDL and LDL cholesterol levels in the serum to risk management in atherogenic disease, considerable attention has been devoted to testing normal and high-risk individuals for serum levels of HDL, LDL, as well as total cholesterol and triglycerides.
One method for LDL and HDL cholesterol testing is based on the selective precipitation of non-HDL particles in serum by polyanionic compounds, such as dextran sulfate, heparin, and phosphotungstate, in the presence of a group-II cation, such as Mg.sup.2+, Mn.sup.2+, and Ca.sup.2+. The specificity and degree of precipitation are dependent on a variety of factors, including the type and concentration of the polyanion/metal agent. In general, the order of precipitation of serum cholesterol particles, with increasing concentration of polyanion is VLDL, LDL, and HDL. HDL generally remains soluble at concentrations of heparin or dextran sulfate which completely precipitate lower density particles, although minor apoE species of HDL may be coprecipitated with lower density particles.
By selective precipitation of lower density particles, HDL serum cholesterol levels can be determined. The HDL value, along with total serum cholesterol and triglycerides measurements can then be used to calculate LDL according to the relationship: EQU LDL cholesterol=total cholesterol
- triglycerides/5 - HDL cholesterol.
In a typical lipid assay procedure, a small volume of blood is drawn and centrifuged to produce a clear plasma or serum sample fluid. The sample fluid is then aliquoted into several assay tubes, for determination of (a) total serum cholesterol, (b) triglycerides, and (c) HDL cholesterol. The HDL sample is precipitated, as above, and the lower density particles are removed by filtration or centrifugation prior to cholesterol detection. The samples are then reacted with an enzyme mix containing cholesterol esterase, cholesterol oxidase, peroxidase and a dye which can be oxidized to a distinctly colored product in the presence of H.sub.2 O.sub.2. The tubes may be read spectrophotometrically, and the desired total, HDL and LDL cholesterol values determined.
Despite the accuracy and reliability which can be achieved with the liquid-phase cholesterol assay just described, the assay has a number of limitations for use in widespread screening. First, the method uses a venous blood sample, requiring a trained technician to draw and fractionate the blood sample, and aliquot the treated blood to individual assay tubes. At least one of the sample tubes (for HDL determination) must be treated with a precipitating agent, and further processed to remove precipitated material. Although some of these procedures can be automated, analytical machines designed for this purpose are expensive and not widely available outside of large hospitals.